Salicylaldehyde

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 May 2021 to 11 July 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to the OECD TG 474 and in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

July 21, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Salicylaldehyde

EC Number:

201-961-0

EC Name:

Salicylaldehyde

Cas Number:

90-02-8

Molecular formula:

C7H6O2

IUPAC Name:

2-hydroxybenzaldehyde

Test material form:

liquid

Details on test material:

- Physical state: colorless or pale yellow clear liquid
- Purity: 99.5 %
- Supplier: unknown　
- Lot/batch No.: VPMQB

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool location (in a refrigerator, permissible window: 1°C to 10°C, measured values: 3°C to 8°C), light-shielded, airtight
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability of the test substance was confirmed as a result of stability confirmation performed by the study contractor after the completion of the animal study.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Slightly soluble in water (17 g/L, 86°C)
- Reactivity of the test material with the incubation material used (e.g. plastic ware): none reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none
- Preliminary purification step (if any): none

Test animals

Species:

mouse

Strain:

ICR

Remarks:

Crlj:CD1(ICR)SPF

Details on species / strain selection:

Mice are used widely in micronucleus tests, and the strain of mice used in this study was selected because of the properties being well known and the abundance of background documents.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 31.7 to 37.0 g for males and 22.9 to 25.5 g for females in the preliminary test, and 31.0 and 35.2 g for males in the main test.
- Assigned to test groups randomly: under following basis: The animals were stratified by body weight on the day of group allocation (day of first dose) and divided into groups so that the mean body weight in each group was as uniform as possible. Animal assignments were performed by a combination of the computerized block placement method and the random sampling method (necessary groups were formed by block placement method and the test groups and the animal numbers within the group were randomly allocated).
- Fasting period before study: none
- Housing: individually in plastic cages (W155×D245×H150 mm: Clea Japan, Inc.) with bedding (White Flake: Charles River Laboratories Japan, Inc.).
- Diet (e.g. ad libitum): free access to solid feed CRF-1 [Oriental Yeast Co., Ltd., lot No.: 100203 (preliminary test) and 100302 (main test)
- Water (e.g. ad libitum): free access (Gotemba City Waterworks, water bottle used)
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C to 23°C for the preliminary test and 20°C to 23°C for the main test
- Humidity (%): 48% to 54% in the preliminary test and 48% to 56% in the main test
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: May 26, 2010 / To: June 25, 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Since the test substance is a type 3 petroleum, corn oil was selected as it allows preparation of satisfactory solution for administration.
- Concentration of test material in vehicle: 12.5; 25; 50 and 100 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw (total volume administered)
- Lot/batch no. (if required): PEG0519
- Purity: for biochemistry

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each concentration was prepared by weighing the test substance and diluting by adding corn oil to make the specified volume.
- storage method: The amount required for each day was dispensed into brown glass bottles and stored in a cool location [in a refrigerator, permissible window: 1°C to 10°C, measured values: 4°C to 5°C (preliminary test), 3°C to 5°C (main test)], and then used within eight days of preparation.
- stability: The solutions at the concentration of 0.1 mg/mL and 200 mg/mL have been confirmed to be stable for up to eight days in a cool location (in a refrigerator) and 24 hours at room temperature according to the confirmation by BoZo Research Center Inc.
- confirmation of the concentration of the test solution: Each concentration of the test solution used in the main test was sampled in the amount of 10 mL each for the confirmation of concentration. The result showed that the percentage of concentration to the labeled value of each concentration was 103.6% to 105.0%, which was within ±10% when labeled value was set as 100, indicating no problems in the result.

Duration of treatment / exposure:

2 days

Frequency of treatment:

Twice, at about 24 hours interval

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Preliminary and main test

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Preliminary and main test

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Preliminary and main test

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Preliminary test

Doses / concentrations 5

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Preliminary test

No. of animals per sex per dose:

3M+3F for the preliminary test.
5M for the main study.

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Justification for choice of positive control(s): This was selected according to the Toxicity Guidelines as MMC is used widely in micronucleus test and abundant background data are available.
- Route of administration: Oral (gavage)
- Doses / concentrations: 1 mg/kg bw/day - 0.1 mg/mL

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
The specimen for micronucleus observation was prepared according to the method by Schmid. The mice applicable to the specified time after administration were euthanized by cervical dislocation, and then both femurs were extracted and amputated at both ends. Following this, the bone marrow cells were extracted into a centrifuge with about 0.1 to 0.2 mL of fetal calf serum (GIBCO BRL, lot No. 494548) using 1 mL disposable injection syringe and 23 G injection needle. The bone marrow cells and the fetal calf serum were mixed with this injection syringe and needle to make the cells loose, and then centrifugation (Tomy Industries, Co., Ltd., tabletop multi-tube centrifuge LC-220) was performed at 1000 rpm for 5 minutes. Following this, the supernatant was discarded, and the precipitate was mixed well in a mixer before smearing onto a slide glass (one slide of specimen was prepared for each of the left and right femur in each animal). The smeared specimen was air dried and fixed with methanol (Wako Pure Chemical Industries, Ltd., lot No.: KWP4454) for 3 minutes and then air dried again. For the preparation of specimens, the comparison table of the animal number and of the sample number was prepared, and the specimen was attached with labels displaying the study number, stage, sample number, type of test, and date of preparation (blinding).

METHOD OF ANALYSIS:
One slide glass with satisfactory smear conditions was selected based on the sample number as the observation is performed under blinded conditions. Fluorescent staining of the bone marrow smear sample with acridine orange and the observations were performed according to the method by Hayashi et al. A slide was placed on a cover glass with a small amount of 40 µg/mL acridine orange solution dropped beforehand, and the observation was performed at the total magnification factor of 600 using a fluorescent microscope (system biological microscope BX40: Olympus Corporation, universal reflected light fluorescence device BX-FLA: Olympus Corporation) with the transmission of excitation light near the wavelength of 489 nm and wavelength of 515 nm as the observation filter. The specimens were discarded after the completion of observations, and the specimens that were not used for observations were discarded after the observation of all specimens.

Evaluation criteria:

For each animal, the number of PCE in 200 total erythrocytes and the number of MNPCE in 2000 PCE were counted, and the incidences (%) were calculated. In addition, the mean and standard deviation were calculated for the number of MNPCE and incidence (%) and the number of PCE and incidence (%) in each group with the maximum and minimum values also calculated for the incidences (%).

Statistics:

Significance for the incidence of micronucleus was determined by first confirming that the incidence (%) of MNPCE in the negative and positive control groups are within the range of mean ± 3 SD of the respective background data at the laboratory, and then the negative control group and the test substance groups were compared by the Kastenbaum-Bowman test based on binomial distribution (significance level: one-sided 5%) and the Cochran-Armitage test for trends (significance level: two-sided 1% and 5%). For the incidence of PCE, the negative control group and each test substance group were compared by Bartlett’s test to investigate the equality in variance (significance level: two-sided 1%), and then Dunnett’s test (significance level: two-sided 1% and 5%) was performed as the variance was found to be equal.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: As a result of oral administration in male and female mice at the doses of 125, 250, 500, 1000, and 2000 mg/kg per day twice at the interval of about 24 hours in the preliminary study, all male and female animals in the 1000 and 2000 mg/kg per day groups were found to have died by the day after the second dose. Animals with decreasing tendencies in body weight were observed for all groups. Based on the above, the dose for the main test was set as follows: 500 mg/kg per day, which was the dose without deaths and was thought to be the maximum tolerable dose, was set as the high dose; this dose was divided by the common ratio of 2 to set the doses of 250 and 125 mg/kg per day, resulting in three doses.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity: No changes in general conditions were observed in the test substance groups, and no changes in generation conditions were observed in the negative control group and the positive control group. No abnormalities were found in the changes in the body weight of animals in the test substance groups or the positive control group in comparison with the negative control group.
- Ratio of PCE/NCE (for Micronucleus assay): In the test substance groups, the incidence of MNPCE was found to be 0.09% ± 0.08% for the 125 mg/kg per day group, 0.09% ± 0.05% for the 250 mg/kg per day group, and 0.11% ± 0.02% for the 500 mg/kg per day group. Compared with 0.07% ± 0.08% in the negative control group, no statistically significant increase was observed for any test substance group, and no dose-dependent changes were observed. In addition, the incidence of PCE in 200 total erythrocytes for the test substance group showed no statistically significant changes compared with the negative control group. Meanwhile, the incidence of MNPCE in the positive control group was found to increase notably compared with the negative control group, and the incidence of MNPCE in the negative and positive control groups were within the range of mean ± 3 SD of the respective background data at the laboratory.
- Appropriateness of dose levels and route: yes, administration in the main test was performed at the high dose of 500 mg/kg per day, which was the dose without deaths in the preliminary test, as well as 250 and 125 mg/kg per day, as an oral dose twice at the interval of about 24 hours.
- Statistical evaluation: No statistically significant increase was observed for the incidence of MNPCE in the test substance groups.
- Other: the main test was performed for male animals only as no notable difference was observed in the onset of toxicity between the sexes.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test item did not induce chromosomal aberrations in the bone marrow of Crlj:CD1(ICR)SPF mice and is therefore not considered to be a clastogenic or aneugenic substance.

Executive summary:

In an in vivo mammalian cell cytogenetics assay (micronucleus) performed according to the OECD Test Guideline No. 474 and in compliance with GLP, the test item diluted in corn oil was administered orally to Crlj:CD1(ICR)SPF mice at the doses of 125, 250, and 500 mg/kg bw per day twice at the interval of about 24 hours, and then a bone marrow smear specimen was prepared at about 24 hours after the second dose. In addition, negative and positive control groups were with a single administration of corn oil and 1 mg/kg mitomycin C, respectively.

 

In the preliminary test performed to determine the doses, the test substance was administered orally to mice at the doses of 125, 250, 500, 1000, and 2000 mg/kg per day twice at the interval of about 24 hours, and as a result all male and female animals in the 1000 and 2000 mg/kg per day groups were found to have died by the day after the second dose. Animals with decreasing tendencies in body weight were observed for all groups.

Based on the result obtained, administration in the main test was performed at the high dose of 500 mg/kg per day, which was the dose without deaths in the preliminary test, as well as 250 and 125 mg/kg per day, as an oral dose twice at the interval of about 24 hours. The bone marrow was collected about 24 hours after the second dose. In addition, corn oil was administered at the same interval as the test substance for the negative control group, and 1 mg/kg mitomycin C was administered once for the positive control group. As a result of the test, no statistically significant increase was observed for the incidence of MNPCE in the test substance groups in comparison with the negative control group, and no dose-dependent changes were observed. In addition, the incidence of PCE in 200 total erythrocytes for the test substance group showed no statistically significant changes compared with the negative control group. Furthermore, the incidence of MNPCE in the negative and positive control groups were within the range of mean ± 3 SD of the respective background data at the laboratory; therefore, the test was thought to be conducted appropriately.

  

Under the test conditions, the test item did not induce chromosomal aberrations in the bone marrow of Crlj:CD1(ICR)SPF mice and is therefore not considered to be a clastogenic or aneugenic substance.

 

 This study is classified as acceptable and satisfies the requirement for an in vivo cytogenetic mutagenicity assay.